Endogenous megakaryocytic colony formation and thrombopoietin sensitivity of megakaryocytic progenitor cells are useful to distinguish between essential thrombocythemia and reactive thrombocytosis.

Diagnosis of essential thrombocythemia (ET) is controversial and remains mainly an exclusion diagnosis. Endogenous megakaryocyte colony (EMC) formation have been largely evaluated to identify specific criteria for ET, but results are impeded by the lack of medium standardization. We evaluated megakaryocyte (MK) colony formation in a serum-free collagen-based medium, without cytokine and in the presence of various concentrations of thrombopoietin (TPO). Thirty-six bone marrows from patients diagnosed with ET (n = 11), polycythemia vera (PV; n = 12), reactive thrombocytosis (RT; n = 6) and healthy donors (n = 7) were assessed. We demonstrate that 11 out 11 of the ET patients had spontaneous megakaryocyte colony-forming unit (CFU-MK) formation, in contrast to none of the RT patients and healthy donors. MK progenitors from ET patients remained responsive to TPO, because exogenous addition of TPO significantly increased cloning efficiency. Moreover, at low doses of TPO (0.5 ng/ml and 5 ng/ml), the number of positive cultures and mean number of TPO stimulated CFU-MK were significantly higher in cultures of cells from patients with ET than in patients with RT. In summary, we have described a standardized serum-free, collagen-based assay that allows differential diagnosis of ET and RT, according to endogenous CFU-MK formation and sensitivity to TPO.

Deletion 7q in B-cell low-grade lymphoid malignancies: a cytogenetic/fluorescence in situ hybridization and immunopathologic study.

Ten cases presenting a simple karyotype and del(7q) as a primary event were selected out of 353 patients referred as B-cell low-grade malignant lymphoproliferative disorders. Chromosome 7-specific painting probes confirmed the deletion that was tentatively assigned to bands q31q35. Chromosome 7 was involved in an interstitial deletion in seven cases, in an unbalanced translocation in two cases, and in a ring chromosome in one case. Common clinical/hematological features included advanced age, marked splenomegaly, and peripheral blood monoclonal IgM(D) lymphocytosis. Regardless of morphologic entity, most cases shared lymphoplasmacytoid features. Deletion 7q may delineate a variety of low-grade B-cell lymphoid disorders characterized by a common clinical history and immunopathologic similarities. The cytogenetic pattern and the ongoing work on molecular mapping of this deletion suggest that the loss of a putative tumor-supressor gene at 7q31q32 may constitute an early event in their pathogenesis.

Effects of two sex steroids (17beta estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937.

We investigated the effects of two sex steroids (17beta estradiol and testosterone) on five human leukemia cell lines. We observed a statistically significant inhibition of proliferation, dose and time dependent, of the human monoblastic leukemia cell line U937. This inhibition was associated with a dose dependent decrease in the number of CFU-blasts in clonogenic cultures. Cytostatic effect was obtained with doses of 5 microM for estrogen and 10 microM for androgen and was not due to a non-specific cytotoxic effect, some cell viability remained high (> 90%) even after 6 days of incubation. More accurately, we demonstrated that growth inhibition was associated with a cell cycle arrest, U937 cells accumulating in G2/M phase. This blockade was dose related with a maximum number of cells accumulating at day 4. Sensitivity of these cells to an S-phase specific agent (hydroxyurea) was not increased, suggesting that these cells were blocked in G2/M and did not undergo mitosis. Expression in U937 cells of high affinity nuclear receptors for estrogen and androgen was negative which was in favour of a type II estrogen binding site, mediated mechanism. Moreover, a small fraction of these cells underwent apoptosis or differentiation with about 12% apoptotic cells and a significant increase (more than 30%) of two myelomonocytic markers (CD13 and CD64). These results demonstrate that the proliferation of some leukemic cells may be inhibited by micromolar concentrations of sex steroids, independently of nuclear receptor expression. The main mechanism seems to be a block in cell cycle associated with modulation of apoptosis and differentiation. It provided additional evidence for the potential value of sex steroids and their analogues in the treatment of leukemias.

Phenotypic, morphologic changes and Ig secretion induced on B-NHL cells in vitro by interferon alpha and all-trans-retinoic acid: possible progression toward a more differentiated state.

Twenty-five B-cell-nonHodgkin's lymphomas (B-NHL): 6 lymphocytic, 2 centrocytic, 13 follicular, centrocytic/centroblastic, 2 lymphoplasmocytoid and 2 centroblastic were tested for their ability to acquire features of mature plasma cells under the effect of interferon alpha (final concentration, 600 UI/ml), all-trans-retinoic-acid (ATRA) (final concentration, 10(-6) mol/l) and the association of both. B-NHL cells were negatively purified (>99%) by an immunomagnetic method, cultured for 7 d with or without interferon and ATRA, then stained with anti-CD19, CD20, surface Ig, DR, CD38 and with anti-CD138 (syndecan-1) antibody-recognizing plasma cells. Ig production was estimated in culture supernatants by an ELISA method and changes in cell morphology were investigated on May-Grunwald-Giemsa-stained cytospin preparations. In all cases interferon and ATRA, alone or in association, were able to induce changes in the immunophenotypic profile, associated or not with morphologic changes and induction of Ig secretion. All changes were greatly variable from one to the other B-NHL sample and no relationship could be found between a particular pattern of change and the histological subtype. Interferon alpha was more potent than ATRA in inducing changes. In favour of a differentiation process, we observed a concomitant decrease of DR expression and increase of CD38 expression in 8 cases with interferon alpha, and in 4 cases with ATRA. Although interferon- or ATRA-treated cells did not display cytologic, functional features and changes of the immunophenotypic profile fully compatible with those of terminally differentiated cells, these results suggest a possible transition toward more differentiated elements, especially with interferon alpha.

Autotumour reactive T-cell clones among tumour-infiltrating T lymphocytes in B-cell non-Hodgkin's lymphomas.

Seventy-three T-cell clones (TCC) were established from tumour-infiltrating lymphocytes-T (TIL-T) derived from lymph nodes involved by B-cell non-Hodgkin's lymphomas (B-NHL) in nine patients with different histological subtypes and clinical stages. 40 TCC (55%) expressed the CD25 Ag and were also able to proliferate in the presence of irradiated autologous B-NHL cells. Among them, 23 autotumour (AuTu) proliferative TCC were found not to proliferate to autologous EBV-transformed B-cell lines, indicating that the proliferative reactivity of these TCC was preferentially directed at autologous B-NHL cells. Tested against autologous B-NHL cells, only three AuTu proliferative TCC (CD8+) showed a significant level of cytotoxicity (specific lysis > 15%). In blocking experiments, the AuTu proliferative reactivity of three TCC from one patient was strongly inhibited by anti-DR and anti-DQ mAbs, whereas that of three TCC from another patient was not affected by either anti-MHC class I or class II (DR, DP, DQ) mAbs. These findings suggest that the recognition of autologous B-NHL cells by AuTu proliferative TCC may occur through MHC-restricted as well as MHC-unrestricted mechanisms.

Accumulation of T-cell clones producing high levels of both granulocyte-macrophage and macrophage colony-stimulating factors (CSF-1) in lymph nodes involved by Hodgkin's disease.

We have previously shown that total T cells derived from lymph nodes (LN) involved by Hodgkin's disease (HD) secrete higher levels of colony-stimulating activity than total T cells present within benign hyperplastic (BH) LN and B-non-Hodgkin's lymphoma (B-NHL) LN, suggesting that T cells with particular properties accumulate in HD LN. To further characterize this T-cell population, we have quantified production of both granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) production in a total of 98 T-cell clones (TCC) derived from CD25+ activated T cells present in HD LN; TCC derived from CD25+ T cells obtained from B-NHL LN(101 TCC), BH LN(95 TCC), and peripheral blood (PBL; 38 TCC) of healthy donors were used as controls. HD LN were characterized by the presence of an elevated number (44%) of TCC producing particularly high titers of both GM-CSF and M-CSF, whereas only a minority of such TCC was found in control groups (10% in B-NHL, 16% in BH, 8% in PBL). These observations support the hypothesis of a selection of T-cell families with particular properties occurring in contact with Reed-Sternberg (RS) cells. According to the biological properties of GM-CSF and M-CSF, it seems reasonable to suggest the involvement of this particular subset of T cells in the granulomatous process, the peripheral blood polynucleosis, and in the paracrine growth of RS cells.

CD45RA expression by CD4 T lymphocytes in tumors invaded by B-cell non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD).

Little is known about the role of tumor infiltrating T lymphocytes (TIL-T) in the pathogenesis of malignant diseases and collaboration between normal and malignant cells has not yet been proved. In the present work, we have investigated whether immune T lymphocytes exist in tumors invaded by B-cell non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD). Therefore, we have studied the reactivity of the CD45RA monoclonal antibody, which discriminates between naive and memory CD4 T lymphocytes. Our results showed far lower percentages of CD4+ CD45RA+ in malignant lymphoma (30.3 +/- 15.0% in B-cell NHL, and 37.4 +/- 18.6% in HD) than in reactive hyperplasia (54.7 +/- 13.2%), leading to the conclusion of an accumulation of immune cells in tumor microenvironment. A further heterogeneity in the relative proportion of naive and memory TIL-T was also observed within lymphoma (range: 11 to 68% in B-cell NHL, 5 to 69% in HD). In B-cell NHL, it was related to histological features, as documented by the Kiel classification (P = .028), and to a stronger extent to cytological characteristics analysed with the Grenoble classification (P less than .0001): class 1 NHL, which are essentially indolent NHL displayed lower naive cells (22.2 +/- 7.4%) than class 3 NHL, which are more aggressive (40.1 +/- 16.1%). Among the monoclonal antibodies (mAb) defining the B-cell clone phenotype or activation state (CD19, CD20, CD21, CD22, CD23, CD24, CD5, CD10, CD11a, and Ki67), only CD23 (P = .0003) and Ki67 (P = .0007) revealed statistical association with the percentage of naive CD4 lymphocytes. No correlation could be demonstrated with the proportion of whole TIL-T, activated CD3 DR TIL-T, or CD4 subset.

Prognostic value of the epithelial membrane antigen (EMA) in the case of Hodgkin's disease.

The study deals with the results obtained from 155 lymph node biopsies of patients suffering from Hodgkin's disease (HD) who were treated by MOPP and radiotherapy in the same establishment. The specimens in paraffin have been examined for the presence of antigens using several monoclonal antibodies, particularly epithelial membrane antigen (EMA). There exists no correlation between the immunophenotypes towards EMA and histological types. Thirty-three patients whose responses were EMA (+) have the assurance survival more than 10 years on the level 32.4% while those 122 patients with EMA(-) had the same survival on 90% level (p less than 0.001). Thus a new prognostic tool has been found which enables to detect the likely therapeutic failures in the case of Hodgkin's disease.

Production of granulopoiesis-stimulating and -inhibiting activities by T cells associated with malignant cells in lymphomas.

The possible role of T lymphocytes in the formation of granulomatous reactions seen in certain malignant lymphoid tumours was investigated by measuring the granulopoietic colony-stimulating activity (CSA) and granulopoietic-inhibiting activity (IA) produced by stimulated T-lymphocytes isolated from peripheral blood, spleen and lymph nodes of patients and normal subjects. Lymph-node T-cells from patients with benign lymphoid hyperplasia, B-cell non-Hodgkin's lymphoma (B-NHL), and non-granulomatous Hodgkin's disease (HD) showed no CSA, but the cells produced IA of 40 +/- 23%, 40 +/- 24% and 50.5 +/- 22.5% respectively. The corresponding cells from patients with HD accompanied by granulomatous reactions produced CSA of 6.85 +/- 6.5 u/microliters and IA of 23.5 +/- 21%. The presence of a granulomatous reaction in malignant lymphoma was correlated with the stimulation of granulopoiesis in vitro by T lymphocytes associated with malignant cells. A correlation was demonstrated between neutrophilic and eosinophilic colonies obtained in vitro under the influence of CSA-producing T cells isolated from malignant lymphomas and the neutrophils and eosinophils present in the granuloma. These results showed that tumour-infiltrating T cells play a role in the presence of granulomatous reactions seen in lymphomas. Peripheral-blood T cells from healthy subjects, and from patients with B-NHL, or with HD unaccompanied by granulocytic reactions produced CSAs of, respectively, 5 +/- 0.5 u/microliter, 4.8 +/- 2.2 u/microliters and 5.3 +/- 0.4 u/microliters, and IAs of 45 +/- 18%. 50 +/- 5.5% and 50.5 +/- 7% respectively. The corresponding values for HD patients with granulocytic reactions were CSA. 17 +/- 15.5 u/microliters, and IA, 9.5 +/- 9%. No correlation was demonstrated between neutrophilic colonies obtained in vitro under the influence of HD blood T cells and neutrophils present in blood. Only one correlation was found: between the percentage of eosinophilic colonies and the number of blood eosinophils. HD blood T cells did not seem to explain completely granulocytic reactions seen in blood.

T lymphocytes from invaded lymph nodes in patients with B-cell-derived non-Hodgkin's lymphoma: reactivity toward the malignant clone.

Tumor-infiltrating T lymphocytes (TIL-T) are always present in B-cell-derived non-Hodgkin's lymphoma (NHL). In this investigation, we explored the possibility that collaboration might exist between these cells. TIL-T were isolated from 39 lymph nodes of patients with NHL. In most of the cases, few of them (less than 10%) possessed surface activation receptors CD25 or OKT9. In 80% of the cases, they proliferated in response to recombinant interleukin-2 (rIL-2), but the degree of proliferation was often low as compared with control populations. The influence of irradiated autologous malignant cells on the TIL-T proliferation in response to rIL-2 (40 U/mL) was also investigated: in 38% of the cases, this proliferation was not modified (group O), and in 41% it was higher (group +) and in 21% it was lower (group -). The mechanism of this immune response (specific or not) is not elucidated at present. The definition of these groups was statistically correlated with different parameters of the disease: (1) percentage of TIL-T was higher in group + (44% +/- 17%) than in group O (31% +/- 18%) and group - (24% +/- 15%); (2) B-cell proliferation in centrofollicular lymphomas was more frequently nodular or nodular and diffuse in group + (83%) and O (55%) than in group - (0%); (3) low-grade malignancies in the Working Formulation were more frequent in group + (75%) than in group O (60%) or group - (12%); (4) favorable prognosis evaluated with the Grenoble cytologic classification was more frequent in group + and O (87%) than in group - (12%); (5) actuarial survival curves showed a significantly better prognosis for patients in group +.

Non-Hodgkin's malignant lymphomas in patients older than 80. 70 cases.

In two centers (a comprehensive cancer center and a university hospital) 1300 patients with non-Hodgkin's lymphomas (NHL) were observed during the past 15 years. Seventy patients (5.4%) were diagnosed after they were 80 years old. Thirty-four patients had low-grade NHL, eight had intermediate grade, and 28 had high-grade NHL. The majority of them were classified as Stage I (n = 24) or II (n = 18), and the rest as Stage III (n = 16) or IV (n = 12). Treatment varied with grade of NHL, tumoral extension, and performance status. Forty-seven patients were given chemotherapy, 37 radiotherapy, and six patients had surgery. Overall, treatment was considered to be optimal in 12 patients, good in 34 patients, and limited in 24 patients. Toxicity was minimal in 62 patients, treatment-limiting in two patients, and lethal in six patients. Thirty-seven patients achieved a complete remission (CR), 21 a partial remission, five experienced stabilization, and seven failed to respond. For all patients, the median survival (MS) was 18 months. In contrast to younger patients, malignancy grade had no significant influence. The only significant parameter is CR (P = 0.02). In conclusion, very old patients must be treated correctly but carefully. Better tolerated treatments are needed to improve results that are neither very poor nor as good as in younger patients.

Splenectomy during chronic lymphocytic leukemia.

The combination of discontinuous high-dose chlorambucil therapy with splenectomy greatly increased the prognosis of aggressive forms of chronic lymphocytic leukemia (CLL). The median survival for 43 patients was 84 months from diagnosis and 48 months from splenectomy. For 15 stages O, according to Rai classification, obtained after splenectomy, duration ranged from 3 to 105 months. The median survival of a group of patients showing "nodular splenic infiltration" was 104 months and superior to that of a group of patients showing "diffuse splenic infiltration" (72 months). In four of 15 cases studied, the peripheral blood lymphocytic clone disappeared after splenectomy.

[Malignant non-Hodgkin's lymphoma associated with a serum monoclonal immunoglobulin]. / Lymphomes malins non hodgkiniens associés à une immunoglobuline monoclonale sérique.

Among the 394 non-Hodgkin's lymphomas (NHL) observed at our hospital over a thirteen year period, we have found 17 associated monoclonal gammopathies (4.3%). Fourteen gammopathies could be correlated to the lymphoid tumor due to a study of immunological membrane markers and to the comparative evolution of the disease and the monoclonal gammopathy. Two different groups could be distinguished: Six patients out of the fourteen studied (43%) had a gammopathy similar to the surface immunoglobulins of the tumour lymphoid cells. Seven of the seventeen showed, after treatment, a parallel evolution between the tumor and the gammopathy. This originates directly from the tumoral secretion and deserves to be classified among the B lymphoid excretory tumors. Six were IgM and one was IgG. Eight of the fourteen patients studied (57%) did not show any immunological relation between the lymphoma and the monoclonal gammopathy. The evolutions (10 of the 17 patients) of the tumor and of the gammopathy were independent. Among the eleven gammopathies (1 double gammopathy), 5 were IgG, 4 were IgA and 2 were IgM. The incidence of these gammopathies related to the total group tf lymphomas is 2.5%. Given the age of these patients, the association seems fortuitous.

[Non-Hodgkin's malignant lymphoma with cervicofacial expression. Modulation of the radiotherapy-chemotherapy combination according to the cytological class]. / Lymphomes malins non hodgkiniens à expression cervico-faciale. Modulation de l'association radio-chimiothérapique par la classe cytologique.

An analysis was conducted in March 1983, after a mean follow up of 40 months, of cases of cervicofacial stages I and II non-Hodgkins malignant lymphoma in 3 children and 41 adults (mean age: 51 years, range: 6-90 years) treated between 1969 and March 1981. According to the Working Formulation malignancy was low in 4 cases, intermediate in 24 and high in 13; 3 cases could not ne classified retrospectively. Cytologic classification showed 13 of class 1 of low malignancy, 7 of class 2 of high malignancy with leukemic potential, and 16 of class 3 of high malignancy with a course leading to tumor formation. The cavum was involved in 10 cases, the tonsils in 9, the parotids in 1, the uvula in 1, isolated cervical adenopathies in 14, multiple unilateral adenopathies in 3 and bilateral cervical adenopathies in 5 cases. Therapy varied according to the series: in the first series (1969-1975) the 23 cases were treated by radiotherapy alone (40-55 Gy). In the second series (1976-1981) of 21 cases, chemotherapy was given as a function of the cytologic class: prophylactic chemotherapy for 6 months after radiation for classes 1 and 2, initial chemotherapy for 6 weeks, cerebral radiation and methotrexate intrathecally, and maintenance chemotherapy for 3 months in class 3. The failure rate for radiated zones was identical in the 2 series (less than 10%). Adjusted 5-year survival rate was 60% for series 1 against 70% for series 2 (p = 0.9), and adjusted remission rate was 43% against 64% (p = 0.8).

Non-Hodgkin's malignant lymphomas: a cytologic classification based on statistical analysis and correlated with prognosis.

On the basis of cytologic studies of 144 patients with non-Hodgkin's malignant lymphomas (NHML), a cytologic classification was established that was composed of three groups (1, 2 and 3) containing nine classes (1A, 1B, 1C; 2A, 2B, 2C; 3A, 3B, 3C). Statistical analyses were carried out using 52 well-defined elementary cytologic characteristics. All the results given by the data-gathering procedures (hierarchical and dynamic clustering methods), by the descriptive analyses (multifactorial and canonical analysis) and by the stepwise discriminant analysis were in agreement with the proposed cytologic classification system. The most discriminating cytologic characteristics of the classes were the cell size, the nuclear-cytoplasmic ratio, the degree of cytoplasmic basophilia, the homogeneity of the cytoplasm and the number of mitoses. The use of these properties renders this classification system reproducible and applicable to clinical practice. A comparison was made between the cytologic classes and the NHML patients as grouped according to their clinical courses. Of the patients in classes 2A, 2B and and 2C, 94% showed acute leukemic characteristics. Of those contained in classes 3A, 3B and 3C, 78% showed poor prognosis or metastatic patterns. Of those contained in classes 1A, 1B and 1C, 76% showed a good prognosis pattern.